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And C18 Bonded Phases with Peptide Mixtures Conner Mchale, Chuping Luo, Benjamin P

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And C18 Bonded Phases with Peptide Mixtures Conner Mchale, Chuping Luo, Benjamin P Comparison of Phenyl- and C18 Bonded Phases with Peptide Mixtures Conner McHale, Chuping Luo, Benjamin P. Libert, William Miles, Mark Haynes, Stephanie Schuster, Joe DeStefano, and Barry Boyes Presented at ISPPP 2017, Philadelphia Advanced Materials Technology, Inc., 3521 Silverside Rd., Wilmington, DE 19810 Overview Comparison with Peptides and Small Molecules Octadecyl‐silane (ODS,C18) columns are one of the most common bonded‐phases Conditions for reversed‐phase separations of protein and peptide mixtures. In many cases, Column: HALO 160Å, 2.7µm, 2.1x100mm Isocratic: 50‐50 ACN‐Water Peptide Sequence Molecular weight (g/mol) pI resolution between two peaks are critical and need to be distinguished from each K’= 1.42 Flow: 0.5 ml/min Detection: 254 nm Gly‐Tyr GY 238.24 5.98 Temperature: 30 ⁰C other. Mobile phases, organic modifiers, and temperature can help with this Phenyl‐Ethyl Injection Volume: 0.4 µl Val‐Tyr‐Val VYV 379.45 5.98 separation however, different bonded phases may be needed to get the desired Sample Tyr–Tyr–Tyr YYY 507.54 5.97 K’= 1.48 1. Uracil (t0) selectivity and resolution. Three Phenyl‐ silane bonded phases were compared to 2. Phenol Methionine Enkephalin YGGFM 573.67 5.98 3. 4‐Chloro‐Nitrobenzene Leucine enkephalin YGGFL 555.63 5.98 the HALO ES‐C18 phase to see if there are any advantages between them. These Phenyl‐Butyl 4. Naphthalene Angiotensin (1–7) Amide DRVYIHP‐NH 898.02 7.37 superficially porous silica particles have a 160Å pore size, ideal for peptides and 2 Angiotensin II DRVYIHPF 1046.19 7.37 polypeptides up to 20 kDa. Each phase has a unique silane bonding that could K’= 2.47 cause different interactions on the stationary phases. This could show differences Angiotensin 1–12 (human) DRVYIHPFHLVI 1508.79 7.52 in selectivity and resolution and potentially give an advantage in the separation. Phenyl‐Hexyl Neurotensin QLYENKPRRPYIL 1672.92 9.94 Melittin GIGAVLKVLTTGLPALISWIKRKRQQ 2846.50 11.85 K’= 3.53 β‐Endorphin YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE 3465.03 9.05 ES‐C18 Sauvagine XGPPISIDLSLELLRKMIEIEKQEKEKQQAANNRLLLDTI‐NH2 4599.31 5.08 Materials and Methods Bovine Insulin FVNQHLCGSHLVEALYLVCGERGFFYTPKA 5733.49 6.6 Columns of HALO 160Å ES‐C18 and Phenyl phases were produced at Advanced Small molecule testing including neutral compounds shows an increase in Materials Technology, Inc. (Wilmington, DE). SEM images were obtained using a retention as the alkyl chain gets longer, as expected. Zeiss (Jena, Germany) Auriga 60 High Resolution Focused Ion Beam & Scanning Phenyl‐Ethyl Phenyl‐Butyl Peptide Mixture 1 Peak# Ret. Time Width(50%) Tailing F. Resolution(JP) Peak# Ret. Time Width(50%) Tailing F. Resolution(JP) 1 0.745 0.02218 1.3 ‐‐ 1 0.69 0.02028 1.386 ‐‐ Electron Microscope at the University of Delaware (Newark, DE). 2 2.318 0.03185 1.335 34.364 2 1.885 0.03425 1.455 25.866 3 4.056 0.02987 1.235 33.227 3 3.907 0.0324 1.296 35.789 Mobile phase modifiers were obtained from Pierce and Synquest Laboratories. 4 4.711 0.03193 1.23 12.5 4 4.521 0.03387 1.289 10.935 Increase in resolution between critical pairs 5 5.199 0.02932 1.271 9.411 5 5.216 0.03177 1.363 12.5 (TFA/DFA). Acetonitrile was MS grade (JT Baker) and HPLC grade (EMD Millipore). 6 6.789 0.03082 1.119 31.195 6 6.897 0.02997 1.171 32.13 7 8.514 0.02838 1.261 34.375 7 8.641 0.03153 1.422 33.471 Peptides were from Sigma‐Aldrich and AnaSpec. Analytical peptide separations Phenyl‐Hexyl ES‐C18 used the Shimadzu Nexera LC‐30 components (100 µL mixer), with the SPD M30A Phenyl‐Ethyl Peak# Ret. Time Width(50%) Tailing F. Resolution(JP) Peak# Ret. Time Width(50%) Tailing F. Resolution(JP) 1 0.843 0.0235 1.399 ‐‐ 1 1.08 0.02293 1.358 ‐‐ UV detector. An Orbitrap Velos Pro ETD (ThermoScientific, Inc.) was used for MS 2 2.823 0.02868 1.466 44.768 2 3.444 0.0265 1.375 56.437 Phenyl‐Butyl 3 4.569 0.02872 1.332 35.897 3 4.868 0.02745 1.281 31.155 detection. MS was scanned from 300‐2000 m/z; the ESI source was operated at 4 5.238 0.02938 1.349 13.598 4 5.751 0.02905 1.323 18.429 Phenyl‐Hexyl 5 5.595 0.02727 1.359 7.433 5 6.023 0.02637 1.509 5.797 3.5 kV. 6 6.901 0.02923 1.185 27.271 6 7.494 0.03132 1.158 30.092 ES‐C18 7 8.642 0.02963 1.438 34.892 7 9.286 0.02892 1.394 35.103 Conditions Peptide Mixture 1 Column: HALO 160Å, 2.7µm, 2.1x100mm 1. Gly‐Tyr (238.24 g/mol) Mobile Phase: 2. Val‐Tyr‐Val (379.46 g/mol) A: Water/ 0.1% TFA HALO Superficially Porous Particles B: 70‐30 ACN/ Water/ 0.1% TFA 3. Methionine Enkephalin (573.67 g/mol) Resolution between critical pairs also improves with the phenyl‐phases. Certain peptides show different Gradient: 5‐55% B in 10 min. 4. Angiotensin II (1046.20 g/mol) Flow: 0.5 ml/min. 5. Leucine Enkephalin (555.62 g/mol) behaviors on the bonded phases. Especially the Phenyl‐Butyl compared to the Phenyl‐Ethyl. Some Detection: 220 nm 6. Bovine Ribonuclease A (13.7 kDa) Temperature: 60⁰C 7. Bovine Insulin (5733.49 g/mol) peptides are less retained while others are more retained. This could have to do with the different silane Injection Volume: 1.7 µl bondings. (sterically protected vs. tri‐functional) Phenyl‐Ethyl Phenyl‐Butyl Peak# Ret. Time Width(50%) Tailing F. Resolution(USP) Area Peak# Ret. Time Width(50%) Tailing F. Resolution(USP) Area 1 3.365 0.0442 1.255 ‐‐ 416857 1 2.715 0.04488 1.295 ‐‐ 415609 2 3.884 0.05403 1.493 6.257 332155 2 3.362 0.06102 1.644 7.08 330595 Peptide Mixture 2 3 8.533 0.05385 1.6 50.883 293148 3 8.358 0.06278 1.866 46.365 294705 Change in selectivity 4 10.231 0.04725 1.282 19.836 177293 4 10.503 0.05035 1.374 21.975 176731 5 12.661 0.05247 1.5 28.769 346896 5 12.706 0.05883 1.649 23.331 353714 6 14.603 0.052 1.233 21.894 191883 6 14.8 0.05265 1.169 21.882 174198 7 19.194 0.05033 1.311 52.825 188326 7 18.97 0.05493 1.401 45.545 274204 8 19.678 0.0565 1.281 5.34 267025 8 19.463 0.0498 1.363 5.499 199799 Phenyl‐Ethyl Phenyl‐Hexyl ES‐C18 Peak# Ret. Time Width(50%) Tailing F. Resolution(USP) Area Peak# Ret. Time Width(50%) Tailing F. Resolution(USP) Area 1 3.807 0.04032 1.413 ‐‐ 416255 1 4.782 0.03792 1.312 ‐‐ 416091 Phenyl‐Butyl 2 4.865 0.04815 1.65 14.032 329522 2 5.864 0.0441 1.652 15.506 332341 Shell with 160 Å pores 3 9.537 0.05288 1.813 53.785 293167 3 10.502 0.05062 1.817 57.207 302774 4 10.949 0.046 1.415 16.811 172364 4 11.981 0.04527 1.395 18.153 178743 Phenyl‐Hexyl 5 13.324 0.05157 1.686 28.689 347167 5 14.499 0.05087 1.67 30.838 345223 6 15.316 0.04855 1.391 23.316 194021 6 16.852 0.05178 1.261 26.901 168743 The HALO 160Å particle is a superficially porous particle made with a 1.7µm solid core 7 19.333 0.04583 1.512 50.291 195403 7 21.346 0.05012 1.583 51.762 197522 ES‐C18 8 20.087 0.05363 1.454 8.93 271712 8 23.292 0.0604 1.481 20.62 281186 and a 0.5µm porous shell. The shell consists of 160Å pores, ideal for peptides and polypeptides up to 20 kDa. These particles show excellent stability and provide lower Conditions Peptide Mixture 2 overall back pressure when compared to totally porous particles. Column: HALO 160Å, 2.7µm, 2.1x100mm 1. Tyr‐Tyr‐Tyr (507.54 g/mol) Mobile Phase: 2. Angiotensin (1‐7) Amide (898.02 g/mol) A: Water/ 0.1% TFA B: 80‐20 ACN/ Water/ 0.085% TFA 3. Angiotensin II (1046.20 g/mol) Another mix of peptides are examined with the different bonded phases. In this case, Gradient: 5‐55% B in 25 min. 4. Angiotensin (1‐12) human (1508.79 g/mol) Flow: 0.5 ml/min. 5. Neurotensin (1672.92 g/mol) resolution between critical pairs decreases. However, the Phenyl‐Butyl phase shows a Detection: 220 nm 6. β‐Endorphin (3465.03 g/mol) Temperature: 60⁰C 7. Sauvagine (4599.31 g/mol) change in selectivity between the last two peptides of the mix. (sauvagine and melittin) Injection Volume: 2 µl 8. Melittin (2846.46 g/mol) This could be very useful in some cases where peak coelution could be an issue. Analysis of a trastuzumab (IgG1) tryptic digest using Mass Spectrometry Conditions Column: HALO 160Å, 2.7µm, 2.1x100mm Mobile Phase: A: Water/ 10 mM DFA B: Acetonitrile/ 10 mM DFA Gradient: 2‐50% B in 60 min. Flow: 0.5 ml/min. Detection: 220 nm Temperature: 60⁰C k = (t1‐ t0)/t0 α = k2/k1 ∆ α= αphenyl – αc18I Trypsin Digest Sample: Reduced and alkylated mAb was digested at 1:30 protein to enzyme for 4hrs in 50 mM Tris‐HCl (pH 7.8)/1.5M Guanidine‐HCl, followed by formic acid acidification and direct injection.
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